Self-assembly of Severe Acute Respiratory SyndromeCoronavirus Membrane Protein*

Received for publication, June 9, 2009, and in revised form, February 10, 2010 Published, JBC Papers in Press, February 12, 2010, DOI 10.1074/jbc.M109.030270

Ying-Tzu Tseng‡1, Shiu-Mei Wang§1, Kuo-Jung Huang‡, Amber I-Ru Lee§, Chien-Cheng Chiang§,and Chin-Tien Wang‡§2

From the ‡Department of Medical Research and Education, Taipei Veterans General Hospital, and the §Institute of ClinicalMedicine, National Yang-Ming University School of Medicine, Taipei 11217, Taiwan

Coronavirus membrane (M) protein can form virus-like par-ticles (VLPs) when coexpressed with nucleocapsid (N) or enve-lope (E) proteins, suggesting a pivotal role forM in virion assem-bly.Herewedemonstrate the self-assembly and release of severeacute respiratory syndrome coronavirus (SARS-CoV)Mproteinin medium in the form of membrane-enveloped vesicles withdensities lower than those of VLPs formed by M plus N.Although efficient N-N interactions require the presence ofRNA, we found that M-M interactions were RNA-independent.SARS-CoV M was observed in both the Golgi area and plasmamembranes of a variety of cells. Blocking M glycosylation doesnot appear to significantly affect M plasma membrane labelingintensity, M-containing vesicle release, or VLP formation.Results from a genetic analysis indicate involvement of the thirdtransmembrane domain of M in plasma membrane-targetingsignal. Fusion proteins containing M amino-terminal 50 resi-dues encompassing the first transmembrane domain werefound to be sufficient for membrane binding, multimerization,and Golgi retention. Surprisingly, we found that fusion proteinslacking all three transmembrane domains were still capable ofmembrane binding, Golgi retention, and interacting with M.The data suggest that multiple SARS-CoV M regions areinvolved in M self-assembly and subcellular localization.

Coronaviruses are enveloped, positive-strandedRNAvirusesthat cause common respiratory and enteric diseases in humansand domesticated animals (1). They are serologically classifiedas two groups of mammalian coronaviruses plus one groupconsisting of an avian infectious bronchitis virus and a turkeycoronavirus (1, 2). The genome structure and encoded proteinsof the severe acute respiratory syndrome coronavirus (SARS-CoV)3 are similar but not identical to members of the three

classified groups, therefore a new classification has been estab-lished for SARS-CoV (3–6). Coronavirus genome size rangesfrom 27 to 32 kb, the largest among known RNA viruses. Thegene order is 5�-pol-S-E-M-N-3, which encodes viral RNA-de-pendent RNA polymerase and four structural proteins: spike(S), envelope (E), membrane (M, formerly referred to as E1),and nucleocapsid (N) (1, 3).Coronavirus replication occurs entirely in host cell cyto-

plasm, with four structural proteins translated from differentviral RNA transcripts (3). Translated on free polysomes, thehighly basic N (�50–60 kDa) interacts with newly synthesizedviral genomic RNA to form helical nucleocapsids (7). The Mmembrane glycoprotein is translated on membrane-boundpolysomes, inserted into the endoplasmic reticulum (ER), andtransported to the Golgi complex (8, 9). M interacts withnucleocapsids on cellmembranes,most likely at the ERorGolgicomplex (10–14). S and E are also translated on membrane-bound polysomes, inserted into ER, and transported to theGolgi complex. This complex is where E and M proteins inter-act and trigger virion budding, with nucleocapsids enclosed (7,15). S is incorporated into virions via interactions withM. Viri-ons accumulate in large, smooth walled vesicles that eventuallyfuse with the plasma membrane, thus releasing virions intoextracellular spaces (3).Previous studies have demonstrated thatM and E expression

is sufficient for virus-like particle (VLP) formation (16–18),implying that S and N are not essential for coronavirus particleassembly. M is the most abundant coronavirus structural pro-tein, with an expression level in host cells �100-fold greaterthan that of E (18). Except for M proteins in the transmissiblegastroenteritis virus and feline infectious peritonitis virus, bothof which are capable of reaching the plasma membrane (19–21), the M proteins of other coronaviruses (including SARS-CoV M) localize exclusively at the ER/Golgi area, where virusassembly and budding takes place (22–24). Nevertheless, it iswell established thatMplays a key role in directing virus assem-bly and determining viral budding sites (25, 26). Similar to othercoronavirus M proteins, SARS-CoV M spans the lipid bilayerthree times (7). According to recent studies, deletionmutationsinvolving the SARS-CoVM transmembrane domain can affectM subcellular localization (27) and disrupt VLP assembly (28).Although efficient SARS-CoV VLP production requires thecombined expression of M, N, and E (29), M plus N (28, 30) orM plus E (31, 32) have been identified as minimum require-ments for VLP formation. SARS-CoV M has been detected inmedium when expressed alone (32), but the released M mole-

* This work was supported by Grant V96C1-035 from Taipei Veterans GeneralHospital, by Grant NSC95-2320-B-010-041-MY2 from the National ScienceCouncil, Taiwan, Republic of China, and by a grant from the Ministry ofEducation, Aim for the Top University Plan.

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Dept. of Medical Research

and Education, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Road,Taipei 11217, Taiwan. Tel.: 886-2-2871-2121 (ext. 2655); Fax: 886-2-2874-2279; E-mail: chintien@ym.edu.tw.

3 The abbreviations used are: SARS-CoV, severe acute respiratory syndrome coro-navirus; ER, endoplasmic reticulum; VLP, virus-like particle; EGFP, enhancegreen fluorescent protein; �-gal, �-galactosidase; HIV-1, human immunodefi-ciency virus, type 1; GST, glutathione S-transferase; PBS, phosphate-bufferedsaline; MHV, mouse hepatitis virus; wt, wild type; M�CD, methyl-�-cyclodex-trin; MGB, M-�-galactosidase; TEM, transmission electron microscope.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 17, pp. 12862–12872, April 23, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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cules have not been characterized in detail, and the molecularbasis of M secretions has not been elucidated. Furthermore, Mplasma membrane localization remains equivocal. The trans-missible gastroenteritis virus M protein has been described ascapable of reaching the plasmamembrane (19, 20) and of intra-cellular localization (33). Results from one study failed to indi-cate plasma membrane labeling of SARS-CoV M (34), butresults fromanother study indicate that SARS-CoVMis detect-able on cell surfaces as well as in Golgi compartments (35).Here we demonstrate that SARS-CoV M, either tagged or

untaggedwith an EGFP orDsRed fluorescent protein, is detect-able on the plasmamembranes of a variety of cells. Results fromgenetic analyses suggest that the presence of all three trans-membrane domains is necessary for M plasma membranelocalization. Although SARS-CoV M self-assembly involvesboth amino- and carboxyl-terminal regions along the Msequence, amino-terminal 50 residues containing the firsttransmembrane domain are sufficient for conferring M self-association, membrane affinity, and Golgi retention. Thesefindings for SARS-CoV M plasma membrane localization andsecretion in medium indicate an undefined trafficking pathwayin coronavirus assembly and budding.

MATERIALS AND METHODS

Plasmid Construction—Mammalian expression vectors en-coding SARS-CoV M and N were provided by G. J. Nabel (28).A pair of upstream and downstream primers was used toamplify M-coding fragments via PCR-based overlap extensionmutagenesis (36). Two primers were used to introduce a FLAGepitope tag to theM carboxyl terminus, with the SARS-CoVMexpression vector serving as a template: the 5�-GTCTGAGCA-GTACTCGTTGCTG-3� forward primer (referred to as the Nprimer) and the 5�-ATCGGATCCTCACTTGTCGTCGTC-CTTGTAGTCCTGCACCAGCAGGGCGATGTT-3� reverseprimer (containing a flanking BamHI restriction site and FLAGtag-coding nucleotides). Purified PCR product was digestedwith BamHI and EcoRV and ligated into the SARS-CoV Mexpression vector. When constructing a series of M-DsRedfusion expression vectors, the N primer served as the forwardprimer, using the M sequence as a template. Primers used tomake the designated constructs were M-DsRed, 5�-GCGGAT-CCTGCACCAGCAGGGCGATG-3�; M50-DsRed, 5�-CGGG-ATCCAGCTTGATGATGTACAG-3�; M75-DsRed, 5�-CGG-GATCCACCCAGTTGATCCTGTACAC-3�; and M100-DsRed,5�-CGGGATCCCTGAAGCTGGCCACGAAGTA-3�. For M101-DsRed and M160-DsRed cloning, the forward primerswere 5�-CTCTGTCGACCATGCTGTTCGCCAGGACC-AGG-3� and 5�-CTCTGTCGACCATGATCAAGGACC-TGCCCAAGGAG-3� and the reverse primer 5�-GCGGAT-CCTGCACCAGCAGGGCGATG-3�. Amplicons containingSARS-CoV M coding sequences were digested with BamHIand SalI and fused to the amino terminus of pDsRed-Mono-mer-N1 (Clontech). To construct M-EGFP we used the Nprimer (forward) and 5�-GCGGATCCCCTGCACCAG-CAGGGCGATG-3� (reverse). Amplified fragments weredigested and ligated into pEGFP-N2 (Clontech).Both pDsRed-Monomer-Golgi and pECFP-Golgi (Clontech)

encode a fluorescent marker capable of labeling the trans-me-

dial region of theGolgi apparatus. To construct�-galactosidase(�-gal) fusions, we replaced the fluorescent DsRed sequence ineach of the M-DsRed fusion constructs with a �-gal codingsequence derived from an HIV-Gag-�-gal fusion expressionvector (37), yielding M-�gal, M13-�gal, M50-�gal, M75-�gal,M100-�gal, M101-�gal, and M160-�gal. Mutations were con-firmed by restriction enzyme digestion or DNA sequencing.GST-N (formerly named as GST-CoN) has the SARS-CoV Ncoding sequence fused to the carboxyl terminus of GST (38).HIVgpt, a replication-defective HIV-1 expression vector, hasbeen described elsewhere (39).Cell Culture and Transfection—293T,HeLa, or Vero-E6 cells

were maintained in Dulbecco’s modified Eagle’s medium sup-plemented with 10% fetal calf serum (Amersham Biosciences).Confluent cells were trypsinized and split 1:10 onto 10-cmdishes 24 h prior to transfection. For each construct, cells weretransfectedwith 20�g of plasmidDNAusing the calciumphos-phate precipitation method; 50 �M chloroquine was added toenhance transfection efficiency. Unless otherwise indicated, 10�g of each plasmid was used for cotransfection. Culture super-natant and cells were harvested for protein analysis 2–3 dayspost-transfection. For HeLa or Vero-E6 cell transfection, plas-midDNAwasmixedwithGenCarrier (Epoch Biolabs) at a ratioof 1 �g to 1 �l; the transfection procedure was performedaccording to the manufacturer’s protocols.Western Immunoblot—At 48–72 h post-transfection, super-

natant from transfected cells was collected, filtered, and centri-fuged through 2ml of 20% sucrose in TSE (10mMTris-HCl (pH7.5), 100 mM NaCl, 1 mM EDTA plus 0.1 mM phenylmethylsul-fonyl fluoride) at 4 °C for 40 min at 274,000 � g. Pellets weresuspended in IPB (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% TritonX-100, 0.02% sodium azide) plus 0.1 mM phenylmethylsulfonylfluoride. Cells were rinsed with ice-cold phosphate-bufferedsaline (PBS), collected in IPB plus 0.1mMphenylmethylsulfonylfluoride, and microcentrifuged at 4 °C for 15 min at 13,700 � gto remove unbroken cells and debris. Supernatant and cell sam-ples were mixed, with equal volumes of 2� sample buffer (12.5mM Tris-HCl (pH 6.8), 2% SDS, 20% glycerol, 0.25% bromphe-nol blue) and 5% �-mercaptoethanol and boiled for 5 min or(for the M-containing samples) incubated at 45 °C for 10 min.Samples were resolved by electrophoresis on SDS-polyacryl-amide gels and electroblotted onto nitrocellulose membranes.Membrane-boundM,M-FLAG, orM-�gal andHA-Mproteinswere immunodetected using a SARS-CoV M rabbit antiserum(Rockland), anti-FLAG, anti-HA (Sigma), or anti-�-galactosi-dase (Promega) monoclonal antibody at a dilution of 1:1000. ForSARS-CoVNdetection, amousemonoclonal antibody (38)wasused at a dilution of 1:5000. The secondary antibody was asheep anti-mouse or donkey anti-rabbit horseradish peroxi-dase-conjugated antibody (Invitrogen), both at 1:5000 dilution.Laser Scanning ImmunofluorescenceMicroscopy—Confluent

293T, HeLa, or Vero-E6 cells were split 1:80 onto coverslips orLabTek Chambered Coverglass (Nunc) 24 h before transfec-tion. Between 4 and 48 h post-transfection, cells were eitherfixed or directly observed under an inverted laser scanning con-focal microscope (Zeiss Axiovert 200M). For indirect immuno-fluorescence microscopy, cells were washed with PBS and per-

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meabilized at room temperature for 10 min in PBS plus 0.2%Triton X-100 following fixation at 4 °C for 20 min with 3.7%formaldehyde. Samples were incubated with the primary anti-body for 1 h and with the secondary antibody for 30 min. Fol-lowing each incubation, samples were subjected to threewashes (5–10 min each) with Dulbecco’s modified Eagle’smedium/calf serum. Primary antibody concentrations wereanti-SARS-CoV M or anti-�-galactosidase at a dilution of1:500. A goat anti-rabbit or rabbit anti-mouse rhodamine-con-jugated antibody at a 1:100 dilution served as the secondaryantibody (Cappel, ICN Pharmaceuticals, Aurora, OH). After afinal Dulbecco’s modified Eagle’s medium/calf serumwash, thecoverslips were washed three times with PBS and mounted in50% glycerol in PBS for viewing. Images were analyzed, andphotographs taken using the inverted laser Zeiss Axiovert200M microscope.Iodixanol Density Gradient Fractionation—Supernatants

from transfected 293T cells were collected, filtered, and centri-fuged through 2ml of 20% sucrose cushions as described above.Viral pellets were suspended in PBS buffer and laid on top of apre-made 10–40% iodixanol (OptiPrep) gradient consisting of1.25-ml layers of 10, 20, 30, and 40% iodixanol solution pre-pared according to the manufacturer’s instructions (Axis-Shield, Norway). Gradients were centrifuged in an SW50.1rotor at 40,000 rpm for 16 h at 4 °C; 500-�l fractions were col-lected from top to bottom, and densities were measured foreach. Proteins in each fraction were precipitated with 10% tri-chloroacetic acid and subjected to Western immunoblotting.Membrane Flotation Centrifugation—At 48 h post-transfec-

tion, 293T cells were rinsed twice, pelletted in PBS, and resus-pended in TE buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA)containing 10% sucrose and complete protease inhibitor mix-ture. Cell suspensions were subjected to sonication followed bylow speed centrifugation. Post-nuclear supernatant (200 �l)was mixed with 1.3 ml of 85.5% sucrose in TE buffer, placed atthe bottomof a centrifuge tube, and coveredwith a layer of 7mlof 65% sucrose mixed with 3 ml of 10% sucrose in TE buffer.Gradients were centrifuged at 100,000 � g for 16–18 h at 4 °C.Ten top-to-bottom fractions were collected from each tube.Proteins in each fraction were precipitated with ice-cold 10%TCA, rinsed once with acetone, and analyzed by Westernimmunoblot.Coimmunoprecipitation and GST Pulldown Assay—293T

cells transfected with FLAG-tagged M expression vector werecollected in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM

NaCl, 1 mM EDTA, 1% Triton X-100) containing Completeprotease inhibitormixture (RocheApplied Science) andmicro-centrifuged at 4 °C for 15 min at 13,700 � g (14,000 rpm) toremove unbroken cells and debris. Aliquots of post-nuclearsupernatant were mixed with equal amounts of 2� samplebuffer and held for Western blot analysis. Lysis buffer wasadded to the remaining post-nuclear supernatant samples tofinal volumes of 500 �l, and each sample was mixed with 20 �lof anti-FLAG affinity gel (Sigma). GST pulldown protocolswere as previously described (40). Briefly, 500 �l of post-nu-clear supernatant containing complete protease inhibitor mix-ture was mixed with 30 �l of glutathione-agarose beads(Sigma). All reactions took place at 4 °C overnight on a rocking

mixer. Immunoprecipitate-associated resin or bead-boundcomplexes were pelleted, washed tree times with lysis buffer,two times with PBS, eluted with 1� sample buffer, and sub-jected to SDS-10% PAGE as described above.Electron Microscopy—Virus-containing supernatant was

centrifuged through 20% sucrose cushions. Concentratedviral samples were placed onto carbon-coated, UV-treated200-mesh copper grids for 2 min. Sample-containing gridswere rinsed for 15 s in water, dried with filter paper, andstained for 1min in filtered 1.3% uranyl acetate. Excess stain-ing solution was removed by applying filter paper to the edgeof each grid. Grids were allowed to dry before viewing with aJOEL JEM-2000 EXII TEM. Images were collected at30,000� and 60,000�.Cholesterol Quantification—Total cholesterol in isolated

membrane flotation fractions were quantified by fluorometricassay using a cholesterol/cholesterol ester quantification kit(BioVision). Briefly, samples were diluted in cholesterol reac-tion buffer (50 �l/well) and mixed with the provided reactionmixture. Fluorescence was measured with a SpectraMax M5microplate reader (Molecular Devices) following incubation at37 °C for 1 h. Cholesterol concentrations based on the gener-ated standard curve were calculated according to the manufac-turer’s instructions.Statistical Analysis—Data are expressed as mean � S.D.

Differences between experimental (mutant) and control (wt)groups were assessed using Student’s t-tests. Significance wasdefined as p � 0.05.

RESULTS

Assembly and Release of SARS-CoV M in the Form of Mem-brane-enveloped Particles—To test its assembly and releasecapability, SARS-CoV M, tagged or untagged with a FLAGepitope, was expressed alone or together with SARS-CoV Nin 293T cells. Harvested culture supernatants were pelletedthrough 20% sucrose cushion and subjected to Western blotanalysis. Consistent with the previous results (28, 40), both Mand N were readily detected in the medium of cotransfectedcells (Fig. 1, lane 5). Notably, substantial amounts of M andM-FLAGwere present in the medium samples without coex-pressed N (lanes 2 and 3), suggesting that the SARS-CoV M

FIGURE 1. Assembly and release of SARS-CoV VLPs. 293T cells were trans-fected with SARS-CoV M, SARS-CoV N, or SARS-CoV M bearing a carboxyl-terminal-tagged FLAG (M-FLAG) expression vector individually or in variouscombinations. At 48 h post-transfection, supernatants and cells were col-lected and prepared for protein analysis as described under “Materials andMethods.” Medium pellet samples (lanes 1– 6) corresponding to 50% of totaland cell lysate samples (lanes 7–12) corresponding to 5% of total were frac-tionated by 10% SDS-PAGE and electroblotted onto nitrocellulose filters.SARS-CoV M and M-FLAG were probed with rabbit antiserum and SARS-CoV Nwas detected with a mouse anti-N monoclonal antibody.

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is capable of release from cells in the absence of other viralcomponents. However, M-FLAG was apparently incapableof efficient association with N, seeing that N was barelydetectable in medium (Fig. 1, lane 6). This may be due to thedisruption of M-N interaction by FLAG tagged carboxyl-terminally. This explanation is compatible with studies dem-onstrating M carboxyl-terminal region involvement in M-Ninteraction in SARS-CoV (28, 41), mouse hepatitis virus(MHV) (14), and transmissible gastroenteritis virus (26). Totest whether released M proteins were membrane-envel-oped, we treated concentrated supernatants from M-ex-pressing cells with protease in the presence or absence ofnonionic detergent. Our results indicate that extracellular Mbecame undetectable following treatment with protease andTriton X-100 (data not shown), suggesting that released Mproteins were enveloped in lipid bilayers.For further confirmation of the presence of extracellular M

and/orNproteins in pelleted particles, we prepared and studiedsupernatant samples using a TEM. Spherical particles (�100nmdiameter) were observed in bothM- andMplusN-cotrans-fected supernatant samples, but not in mock-transfected sam-ples (Fig. 2A). An iodixanol density gradient fractionation anal-ysis was performed to gather additional evidence of differentdensities between particles formed by M or by M plus N. Asshown in Fig. 2 (B–D), M-formed particles had densities of�1.13 g/ml, slightly lower than for VLPs formed by M plus N(1.14 g/ml). Similar results were observed in three independentexperiments, suggesting that, althoughM alone is sufficient forparticle formation, the incorporation of N intoM vesicles facil-itates the formation of tightly packed VLPs.Glycosylation, Lipid Rafts, and RNA Are Not Required or

Involved in the Self-assembly and Release of M Proteins—Be-cause M protein contains a single N-glycosylation site at thefourth amino acid residue Asn (35), we tested whether glycosy-lation is required for M release. Cells were transfected with aglycosylation-defective M expression vector in which Asn-4was replaced byGln.We also tried to determinewhether SARS-CoV VLP assembly and release involves a cholesterol-enrichedlipid raft-like membrane domain by treating transfectants withthe cholesterol-depletion chemical M�CD. Our results indi-cate that released levels of M, either expressed alone or coex-pressed with N, were not significantly affected by blocking gly-cosylation (data not shown). Surprisingly, quantities of releasedM increased markedly following M�CD treatment (Fig. 3A).However, virus production byHIV-1, whose assembly and bud-ding is lipid raft-dependent, was noticeably reduced by M�CD(Fig. 3B, lane 8), a finding that is consistent with a previousreport (42). Similar results were observed across several inde-pendent experiments. Increased quantities of released M as aresult of cytolysis were minimal (if any), because no gross cyto-toxicity was observed. Furthermore, in the absence of M coex-pression, N was undetectable in medium following M�CDtreatment (data not shown), supporting the proposition thatincreased M release is not a result of cytolysis. Because M par-ticles released fromM�CD-treated cells may be assembled dif-ferently than M particles from control cells, we therefore per-formed additional experiments to determine whether Mreleased fromM�CD-treated cells are assembled in particulate

form similar to M from control cells. We centrifuged M-con-taining supernatants from M�CD-treated or untreated cellsthrough a 20% sucrose cushion. Aliquots of the resuspendedpellets were studied using a TEM. Remaining resuspensionswere centrifuged with M-FLAG particles (concentrated fromthe supernatants of M-FLAG-expressing cells and serving asa control for sampling bias from gradient to gradient)through the same iodixanol gradient. Our results indicatethat the majority of M from either M�CD-treated oruntreated cells co-sedimented with M-FLAG at the samefraction (fraction 4), with a buoyant density between 1.12and 1.13 g/ml (Fig. 3C). Similar results were observed inrepeat independent experiments.

FIGURE 2. SARS-CoV VLP analysis. 293T cells were transfected with M orcotransfected with M and N expression vectors. At 48 h post-transfection,culture supernatants were collected, filtered, and pelleted through 20%sucrose cushions. Pellets were resuspended in PBS buffer, stained, andobserved with a TEM (A). Bars, 200 nm. For buoyant density gradient analysis,concentrated supernatants derived from M (C) or M plus N (B) transfectionsamples were centrifuged through a 10 – 40% iodixanol gradient for 16 h. Tenfractions (equal quantities) were collected from top to bottom. Fractiondensities were measured and SARS-CoV M and N proteins analyzed byWestern immunoblotting probed with anti-M and anti-N antibodies. Mproteins in each fraction were quantified by scanning immunoblot banddensities. Relative M protein level in each fraction was plotted against theiodixanol density (D).

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Our TEM observations indicate that M particles releasedfrom M�CD-treated cells retain membrane integrity andexhibit spherical morphology that is barely distinguishablefrom the M particles released from untreated cells (data notshown). This is in agreement with previous reports indicatingthat M�CD treatment does not significantly affect virion mor-phology (43, 44). However, we cannot rule out the possibilitythat a failure to detect membrane-damagedM particles may bedue to particle instability. It is likely that the fragility of M par-ticles (lacking other viral components such as genomic RNA orthe viral structural proteins S, E, and N) may have caused themto break up following their release from M�CD-treated cells,making membrane-defective M particles (if any) barely detect-able in pellets. Overall, our results suggest that M recoveredfromM�CD-treated cells are assembled in the samemanner asM from control cells and that lipid rafts are not required for Mself-assembly and release. Further studies are required to deter-mine the underlying molecular basis of the M�CD enhance-ment effect on M release.

Based on previous studies suggesting thatM protein in coro-naviruses also possesses an RNA-binding property (45, 46), welooked at whether the presence of RNA is required for SARS-CoV M-M and/or M-N interaction. M or N was coexpressedwith M-FLAG or GST-N, the latter with GST tagged at the Namino terminus. M or N association with M-FLAG or GST-Nwas assessed by coimmunoprecipitation or a GST pulldownassay in the presence or absence of RNase. We previouslyreported that (a) N is capable of undergoing self-association,and (b) its associationwithhumanAPOBEC3G(hA3G) isRNA-dependent (38, 40). GST-N association with hA3G served as acontrol. We observed that equivalent amounts of M werecoprecipitatedwithM-FLAG (Fig. 3D, lane 15) under an RNasetreatment condition of either significantly reduced levels of co-pulled-down N (Fig. 3D, lane 13), or the elimination or near-elimination of co-pulled-down hA3G (Fig. 3E, lane 5). TheRNase treatment did not significantly impact M associationwith GST-N (Fig. 3, D and E). GST by itself was not capable ofpulling down M, N, or hA3G (data not shown). To further

FIGURE 3. Effects of M�CD or RNase A treatment on M release and M-M or M-N interaction. A and B, 293T cells were transfected with a replication-defectiveHIV-1 vector, HIVgpt (B) or cotransfected with SARS-CoV M and SARS-CoV N (A). At 18 h post-transfection, transfectants were split equally onto three dish plates,which were left untreated or treated with 5 or 20 mM of M�CD at 37 °C for 30 min. Cells then were washed twice with PBS and refed with medium. At 2 hpost-medium replacement, cells and supernatant were harvested for Western immunoblot analysis. HIV-1 capsid proteins were detected with an anti-p24gag

monoclonal antibody. Positions of HIV-1 Gag proteins Pr55, p41, and p24 are indicated. C, buoyant density gradient analysis of M particles released fromM�CD-treated cells. Supernatants from SARS-CoV M-expressing 293T cells that were untreated or treated with M�CD (20 mM) as described above werecollected, filtered, and pelleted through 20% sucrose cushions. Pellets were resuspended in PBS and centrifuged with M-FLAG pellets through the sameiodixanol gradient as described in the Fig. 2 legend. Each fraction was measured for density and analyzed for M and M-FLAG protein level by immunoblotting.Asterisks indicate the M-FLAG position. D–F, 293T cells were cotransfected with the designated plasmids. The construct hA3G is an HA-tagged humanAPOBEC3G expression vector. At 48 h post-transfection, equal amounts of the cell lysates were treated with or without 0.2 mg/ml DNase-free RNase A for 30min at 25 °C, followed by mixing with glutathione-agarose beads, anti-FLAG, or anti-HA affinity gel. Complexes bound to the beads were pelleted, washed, andsubjected to Western immunoblotting. The bands (with an asterisk indicating the N position) in the bottom panels of F are the result of the incomplete strippingof the previous anti-N probe.

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confirm that RNA is not essential for M-N interaction, we per-formed an additional coimmunoprecipitation experimentusing an M expression vector carrying an amino-terminal HAtag (HA-M). The result indicates that N was still capable ofassociating with M when treated with RNase (Fig. 3F, lane 12).In contrast, RNase treatment abrogates N association withhA3G (lane 11), which is consistent with the GST pulldownassay results (Fig. 3E). Together, these findings suggest that thepresence of RNA is not necessary forM-M orM-N interaction,but it does stimulate efficient N-N interaction.Retention of Amino-terminal 50 Residues Is Sufficient for M

Multimerization and Membrane Binding—To map domainsinvolved in M protein secretion, we engineered a set of M-�-galactosidase (MGB) fusion constructs containing full-lengthM (M-�gal) or various amino- or carboxyl-terminal M codingsequences (Fig. 4C), and tested the ability of each MGB con-

struct to associate with M. We found that M-�gal is also capa-ble of release into medium, although less efficiently than M(data not shown). Equilibrium centrifugation analysis indicatesthat the majority of M-�gal was sedimented at fraction 6 withan iodixanol density of 1.15 g/ml, higher than that of M parti-cles in the same gradient (Fig. 4A). M andM-�gal coexpressionresulted in peaking M and M-�gal release at the same fractionandwith a density similar to that ofM-�gal (Fig. 4B), suggestingefficient interaction between the two molecules. The higherdensity of M-�gal particles compared with M particles may beexplained, at least in part, by their higher molecular weight.Another possible explanation is that the fused �-gal proteininduced a global conformational change, resulting in M-�galmolecules packed in a more compact manner. Although thischimeric particle assembly system might provide a convenientassay with which to determine required M sequence bound-

FIGURE 4. SARS-CoV M association with M-�gal fusion proteins. A and B, incorporation of M-�gal into M particles. 293T cells were transfected with M orM-�gal expression vector alone or in combination (B). Two days after transfection, supernatants were collected and pelleted through 20% sucrose cushions.Pellets were resuspended in PBS buffer and centrifuged through 10 – 40% iodixanol gradients as described in the Fig. 2 legend. To make direct comparison withM particles, M-�gal pellets were pooled with M pellets and centrifuged through the same gradient (A). Each fraction was measured for density and analyzed forM and M-�gal protein level by immunoblotting. C, schematic representations of SARS-CoV M deletion mutations. Indicated is wild-type (WT) SARS-CoV Mprotein with the three predicted transmembrane domains (shaded boxes). Carboxyl- or amino-terminal residue positions in the deleted mutations were usedto designate the constructs (deleted condons are in parentheses). Dashed lines indicate deleted sequences. Each construct was carboxyl-terminally tagged witha �-galactosidase or DsRed coding sequence. D, coimmunoprecipitation of M-�gal fusion proteins with M-FLAG. 293T cells were cotransfected with M-FLAGand pBlueScript SK or M-�gal fusion construct as indicated. Cell lysates were subjected to Western immunoblotting 48 h post-transfection. Equal amounts ofcell lysates were mixed with anti-FLAG affinity gel for 2 h at 4 °C. Bead-bound complexes were pelleted, washed, and subjected to Western immunoblotting.

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aries for M-M interaction, MGB signals were often barelydetectable following iodixanol density gradient fractionation.We therefore used a coimmunoprecipitation experiment tomap the domain involved inM self-association.M immunopre-cipitation demonstrated interaction with MGB moleculesretaining theM transmembrane domains (Fig. 4D, lanes 14 and16–18). Similar results were observed when the preclearedlysates of individually expressed M-FLAG and MGB weremixed prior to immunoprecipitation (data not shown). Thesedata suggest that efficientMmultimerization is largely depend-ent on the triple transmembrane-domain region. Specifically,amino-terminal 50 residues encompassing the first transmem-brane domain were found to be sufficient for effective M-Minteraction.Next, we performed membrane flotation experiments to

determine whether deleted M sequences exert any effect onMGB membrane binding and if any correlation exists betweenthe multimerization defect and reduced membrane-bindingcapacity. According to our results, �70% of the total cellularM or M-�gal were membrane-associated (Fig. 5); M50-,M75-, and M100-�gal exhibited membrane-binding capaci-ties comparable to or higher than that of M-�gal. AlthoughM100-�gal and M50-�gal are present in higher percentagescompared with M-�gal and M, the differences are not statis-tically significant. In contrast, �10% of total M13- or M160-�gal were membrane-bound. M101-�gal was moderatelydefective in membrane binding (i.e. �50% of total cellularM101-�gal wasmembrane-associated). To confirm the pres-ence of lipid membrane, we quantified cholesterol (a majormembrane lipid component) in each isolated fraction. Themajority of cholesterol was found in the 10–65% sucroseinterface (Fig. 5B), corresponding to the peak fraction (frac-tion 3) of both M and caveolin-1, a known raft-associatedmembrane protein (47). These results suggest that the ami-no-terminal 50 residues bearing the first transmembranedomain are sufficient for conferring efficient membranebinding and indicate a strong correlation between MGBmultimerization efficiency and membrane binding capacity.Additionally, we observed a correlation between MGBrelease efficiency and membrane-binding capacity; in otherwords, MGB fusion proteins considered defective in mem-brane binding (M13-, M101-, and M160-�gal) are ineffi-ciently released (data not shown).To examine whether a correlation exists between M fusion

protein subcellular localization and the above-described mem-brane flotation results, DsRed fusions containing full-lengthM,M13, M50, M70, M100, M101, or M160 sequences were con-structed, expressed in living cells, and analyzed by confocalmicroscopy. We first examined the subcellular distribution ofuntagged M and found that it was primarily localized in theplasmamembrane and perinuclear areas (Fig. 6A). M-DsRed orM-EGFP transfectants (fixed or unfixed) showed fluorescentstaining patterns indistinguishable from those of M transfec-tants (Fig. 6,C–F and L). At 4 h post-transfection,M-EGFPwasmostly found in the perinuclear area and colocalized with theDsRed-Golgi marker (Fig. 6,G–I). Peripheral punctate fluores-cence becamemore pronounced 24hpost-transfection. Similarresults were also observed in Vero-E6 (Fig. 6J) and 293T cells

(data not shown). Combined, these data suggest that SARS-CoV M is capable of targeting the plasma membrane, and thattagged EFGP or DsRed has little (if any) impact onM subcellu-lar localization.We then analyzed domains involved in M localization. Cells

expressing fusions containing M transmembrane domains(M50-DsRed, M75-DsRed, and M100-DsRed) or the carboxyl-terminal half of M (M101-DsRed) expressed enriched fluores-cence around their nuclei (Fig. 6, N–Q). In contrast, cellsexpressing M13-DsRed or M160-DsRed showed diffuse intra-cellular fluorescent staining patterns (Fig. 6, M and R). Results

FIGURE 5. Membrane flotation centrifugation of SARS-CoV M-�gal fusionproteins. A, 293T cells were transfected with the SARS-CoV M, �-gal, orM-�gal expression vectors as indicated. At 2 d post-transfection, cells wereharvested and homogenized. Crude membranes extracted from cell lysateswere subjected to equilibrium flotation centrifugation as described under“Materials and Methods.” Ten fractions were collected from the top down-wards, and fraction aliquots were analyzed by Western immunoblotting. Dur-ing ultracentrifugation, membrane-bound proteins floated to the 10 – 65%sucrose interface. Total M or �-gal-associated proteins were quantified byscanning the immunoblot band densities of the 10 fractions. Percentages ofmembrane-bound proteins were determined by dividing membrane-boundprotein density units (fractions 2– 4) by total protein density units and multi-plying by 100. Mean and standard deviation values for membrane-bound Mor �-gal-associated proteins are indicated. B, 293T cells transfected withSARS-CoV M expression vector were subjected to membrane flotation centri-fugation as described above. Fraction aliquots were analyzed by Westernimmunoblotting and measured for cholesterol level as described under“Materials and Methods.” M and caveolin-1 were probed with anti-M andanti-caveolin-1 antibodies.

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from experiments involving coexpression with a Golgi labelingmarker (pECFP-Golgi) reveal that perinuclear M50-, M75-,M100-, and M101-DsRed localize primarily in the Golgi area(data not shown). These data indicate a correlation between theM sequence involved in membrane binding and Golgi localiza-tion and suggest that amino-terminal 50 residues are sufficientfor M membrane binding and Golgi retention. Surprisingly,M100-DsRed transfectants expressed enriched fluorescence in

both peripheral and perinuclear areas (Fig. 6P), a staining pat-tern similar but not identical to that of M-DsRed. This impliesthat retention of the three transmembrane domains is essentialfor SARS-CoV M plasma membrane localization.Multiple SARS M Regions Are Involved in M-M Interaction—

Although the coimmunoprecipitation experiment results sug-gest that amino-terminal transmembrane regions dictate Mself-association, the possibility that the carboxyl-terminalregionmay also be involved inM-M interaction cannot be over-looked. To gain insight intoM domains involved in self-associ-ation, M-EGFP was individually coexpressed with M-, M13-,M50-, M75-,M100-,M101-, orM160-DsRed, and resulting fluo-rescence distributions were analyzed by confocal microscopy.We reasoned that M-EGFP might dominantly affect DsRedsubcellular distribution patterns; although we could notexclude the possibility of DsRed fusion localization signals con-founding assay results. As expected, colocalization betweenM-DsRed andM-EGFPwas readily observed in the perinuclearand plasma membrane areas (Fig. 7, A–C). Whereas M50-,M75-, and M100-DsRed fusions largely colocalized withM-EGFP, their subcellular distributions were not significantlyaffected by the coexpressedM-EGFP (Fig. 7,G–O, versus Fig. 6,N–P). Little (although visible) peripheral punctate spot fluores-cence was observed in M50- and M75-DsRed cotransfectants.In contrast, M101-DsRed (localized exclusively around cellnuclei when expressed alone) localized with coexpressedM-EGFP to plasma membrane besides the perinuclear area(Fig. 6Q versus Fig. 7, P–R). Although the M160-DsRed trans-fectants expressed a diffuse intracellular fluorescence pattern,significant peripheral punctate fluorescence was only observedin cells cotransfected withM-EGFP (Fig. 6R versus Fig. 7, S–U).These data suggest thatM-EGFP can influence the distributionpattern of M101-DsREd and M160-DsRed, presumablythrough an interaction involving the M carboxyl-terminalregion. These findings support the proposal that SARS-CoVMamino- and carboxyl-terminal regions are both involved in Mself-association.We performed membrane flotation centrifugation experi-

ments to corroborate the involvement of the carboxyl-terminalregion in M-M interactions, with M-FLAG coexpressed witheitherM101- orM160-�gal. BecauseM101- andM160-�gal aremoderately to severely defective in membrane binding, wereasoned that M coexpression would increase fusion proteinmembrane-associated quantities if they are capable of associat-ing with M. We found that M coexpression resulted inincreased quantities of membrane-bound M101-�gal, but at astatistically insignificant level. Membrane-associated M160-�gal quantities increased dramatically following M coexpres-sion, �8-fold compared with M160-�gal expression alone(Figs. 8 versus 5). However, HA-M160 (a membrane-binding-competent M mutant with a deleted carboxyl-terminal se-quence downstream of codon 160), failed to significantlyincrease membrane-associated quantities of M160-�gal. Thesefindings suggest that, even though the M carboxyl-terminalregion is involved in M-M interactions, such interactions areinsufficiently robust to enable M101- or M160-�gal coprecipi-tation with M-FLAG.

FIGURE 6. Subcellular localization of SARS-CoV M (untagged or taggedwith a fluorescent protein) in fixed or living cells. HeLa (A–F and K–R), 293T(G–I), or Vero-E6 (J) cells were transfected or cotransfected with the indicatedexpression vectors. pM-EGFP and pM-DsRed encode SARS-CoV M bearingcarboxyl-terminal-tagged EGFP and DsRed, respectively. pDs-Red-Golgiencodes a Golgi apparatus labeling marker. At 4 h (G–I) or 24 h post-transfec-tion, cells were either fixed or directly observed using a laser confocal micro-scope. Fixed cells (A and D–F) were labeled with a primary anti-SARS-CoV Mantibody and a secondary rhodamine-conjugated anti-rabbit antibody.Images shown here represent the most prevalent phenotypes. Merged redand green fluorescence images (D and E) are shown in F. Superimposed fluo-rescence and phase-contrast images (G and H) are shown in I. Mock-trans-fected cells failed to yield any signal (data not shown).

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DISCUSSION

Findings from previous immunofluorescence studies showthat SARS-CoV M primarily localizes in the perinuclear area(27, 34). Here we demonstrated that SARS-CoV M localizes inboth the plasma membrane and perinuclear areas of 293T,HeLa, and Vero cells. Nal et al. demonstrated that SARS-CoVM-EGFP vesicles traffic out of Golgi compartments in livingBHK-21 cells, with no plasmamembrane labeling detected (34).They proposed that M may retrograde when transported fromGolgi to ER, and/or M may be efficiently endocytosed or recy-cled upon reaching the plasma membrane, resulting in failure

to visualizeM plasmamembrane localization. Accordingly, theSARS-CoVM plasma membrane localization that we observedmay be dependent on cell type.Plasma membrane labeling for M100-DsRed but not for

either M50- or M75-DsRed fusions (Fig. 6) implies that SARS-CoV M may contain a plasma membrane-targeting signalinvolving the third transmembrane domain. Cells expressing aglycosylation-defective M (N4Q) exhibited an immunofluores-cence staining pattern indistinguishable from that of wt trans-fectants (data not shown), suggesting that glycosylation is notrequired for M plasma membrane targeting. Glycosylation isalso dispensable for M self-association and release, as N4Qmutant quantities detected in the medium were near the leveldisplayed bywtM (data not shown). This agreeswith a previousreport that the glycosylation of coronavirus M is not essentialfor MHV VLP assembly (25). Furthermore, the negative effectof the cholesterol-depletion agent M�CD on the release ofM-associated particles was virtually zero (Fig. 3A). This findingis compatible with reports that lipid rafts are required for virusentry but not for virus release inMHV (48) and SARS-CoV (49).Although the presence of RNA is necessary for efficient N-Ninteraction, we found that M-M or M-N interaction does notrequire RNA (Fig. 3). RNA-independent SARS-CoV M-Ninteraction is similar to MHV M-N interaction (13). Despitebeing capable of multimerization, SARS-CoV N was barelydetectable in medium pellets when M plus N VLPs were pre-treated with 0.5% Triton X-100 (data not shown), suggestingthat the formation of high orderNmultimers depends onmem-brane association through N-M interaction. The combinationof M plus N, or of M plus M-�gal, resulted in the formation ofmore dense particles compared with those formed by M alone

FIGURE 7. Subcellular localization of M-DsRed fusion proteins coex-pressed with M-EGFP. HeLa cells were cotransfected with M-EGFP andM-DsRed fusion expression vectors bearing the indicated M mutation. At 18 hpost-transfection, cells were directly viewed using a laser confocal micro-scope. Merged red and green fluorescence images are shown (right-hand col-umn panels). Images represent the most prevalent phenotypes.

FIGURE 8. Membrane flotation centrifugation of M-�gal fusion proteinsin the presence of M. 293T cells were cotransfected with the SARS-CoV Mexpression vector and a �-gal, M101-�gal, or M160-�gal construct, orcotransfected with M160-�gal and an M expression vector carrying an amino-terminal HA tag and a deleted carboxyl-terminal sequence downstream ofcodon 160 (HA-M160). At 48 h post-transfection, cells were harvested andsubjected to membrane flotation centrifugation. Membrane-bound �-galfusion protein percentages were determined as described in the Fig. 5 leg-end. Mean and standard deviation values for membrane-bound �-gal-asso-ciated proteins are indicated.

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(Figs. 2 and 4). This suggests that SARS-CoV M is not a majordeterminant of virus particle density.The possibility thatM-containing particles bud directly from

plasmamembrane cannot be excluded given the capability ofMto localize to plasma membrane. One research team has sug-gested that the coronavirus M protein is responsible for theinduction of � interferon synthesis in leukocytes (50). SARS-CoV M has been shown to be capable of inducing apoptosis inmammalian (51) and insect cells (52). According to a morerecent study, SARS-CoVM is capable of inhibiting type I inter-feron expression by preventing the formation of a TRAF3-TANK-TBK1/IKK (epsilon) complex (53). Because SARS-CoVM is capable of a physical association with TRAF3 (which cantrigger signal transduction following binding to specific plasmamembrane receptors (54)), SARS-CoV M localization toplasma membrane may affect TRF3-mediated signal pathways.It is unknown whether SARS-CoV M released from cells orlocalized at plasma membrane is biologically relevant to theimmune reaction or pathogenesis associated with SARS-CoV(55, 56).As shown in Fig. 5, SARS-CoVMamino-terminal 50 residues

bearing the first transmembrane domain (M50-) are sufficientfor conferring the ability of fused �-gal to efficiently associatewith cell membrane and release. In addition, an effective asso-ciation was noted between M50-�gal and M-FLAG (Fig. 4D),and intracellularM50-DsRedprimarily colocalizedwith aGolgimarker (data not shown). These data suggest that the secondand third transmembrane domains are dispensable for SARS-CoV M Golgi retention, membrane binding, and self-associa-tion. In the case of infectious bronchitis virus, the first trans-membrane domain is both necessary and sufficient for Mlocalization in the Golgi region (8, 57–59). However, all threetransmembrane domains are required for MHVM localizationto the Golgi compartment (60, 61).Our observation that M101- andM160-DsRed (both lacking

the three transmembrane domains) colocalize with M-EGFPon plasma membrane (Fig. 7, P–U), combined with evidenceindicating that full-length rather than truncatedM (HA-M160)coexpression triggers a significant increase in membrane-boundM160-�gal quantities (Figs. 8 versus 5), strongly suggestthe involvement of the SARS-CoVM carboxyl-terminal regioninM-M interaction. This finding differs from those in previousMHVM-M interaction studies demonstrating that the removalof all three transmembrane domains eliminates M-M interac-tion ability (62). Surprisingly, neither M50- nor M75-DsRedeffectively colocalizedwithM-EGFPonplasmamembrane (Fig.7), despite carrying the efficient M-M interaction domain (Fig.4D). One possible explanation is that the Golgi retention signalcontained within M amino-terminal 50 codons becomes thedominant trafficking determinant once the third transmem-brane domain is removed. However, both M101- and M160-�gal are incapable of coprecipitationwithM (Fig. 4D), implyinga membrane association requirement for efficient M-Minteraction.In summary, our data suggest that SARS-CoV M contains a

plasma membrane localization signal involving the third trans-membrane domain. Glycosylation is not required forM plasmamembrane localization, self-assembly, and release. Although

the presence of RNA is necessary for N-N interaction, the sameis not true for M-M orM-N interaction. AlthoughM self-asso-ciation andGolgi localizationmay involvemultipleM sequenceregions, amino-terminal 50 codons bearing the first transmem-brane domain are apparently sufficient for Golgi retention,efficient membrane binding, and SARS-CoV M proteinmultimerization.

Acknowledgments—We thank C. H. Chang and Y. F. Chang forreagents and technical assistance, and Steve S. L. Chen for helpfuldiscussions and suggestions.

REFERENCES1. Lai, M. M. (1987) Adv. Exp. Med. Biol. 218, 7–132. Spaan, W., Cavanagh, D., and Horzinek, M. C. (1988) J. Gen. Virol. 69,

2939–29523. Masters, P. S. (2006) Adv. Virus Res. 66, 193–2924. Ziebuhr, J. (2004) Curr. Opin. Microbiol. 7, 412–4195. Marra, M. A., Jones, S. J., Astell, C. R., Holt, R. A., Brooks-Wilson, A.,

Butterfield, Y. S., Khattra, J., Asano, J. K., Barber, S. A., Chan, S. Y., Clou-tier, A., Coughlin, S. M., Freeman, D., Girn, N., Griffith, O. L., Leach, S. R.,Mayo, M., McDonald, H., Montgomery, S. B., Pandoh, P. K., Petrescu,A. S., Robertson, A. G., Schein, J. E., Siddiqui, A., Smailus, D. E., Stott, J.M.,Yang, G. S., Plummer, F., Andonov, A., Artsob,H., Bastien,N., Bernard, K.,Booth, T. F., Bowness, D., Czub, M., Drebot, M., Fernando, L., Flick, R.,Garbutt, M., Gray, M., Grolla, A., Jones, S., Feldmann, H., Meyers, A.,Kabani, A., Li, Y., Normand, S., Stroher, U., Tipples, G. A., Tyler, S., Vo-grig, R., Ward, D., Watson, B., Brunham, R. C., Krajden, M., Petric, M.,Skowronski, D. M., Upton, C., and Roper, R. L. (2003) Science 300,1399–1404

6. Satija, N., and Lal, S. K. (2007) Ann. N. Y. Acad. Sci. 1102, 26–387. de Haan, C. A., and Rottier, P. J. (2005) Adv. Virus Res. 64, 165–2308. Machamer, C. E., Mentone, S. A., Rose, J. K., and Farquhar, M. G. (1990)

Proc. Natl. Acad. Sci. U.S.A. 87, 6944–69489. Klumperman, J., Locker, J. K.,Meijer, A., Horzinek,M.C., Geuze,H. J., and

Rottier, P. J. (1994) J. Virol. 68, 6523–653410. Hurst, K. R., Kuo, L., Koetzner, C. A., Ye, R., Hsue, B., and Masters, P. S.

(2005) J. Virol. 79, 13285–1329711. Sturman, L. S., Holmes, K. V., and Behnke, J. (1980) J. Virol. 33, 449–46212. Risco, C., Anton, I. M., Enjuanes, L., and Carrascosa, J. L. (1996) J. Virol.

70, 4773–477713. Narayanan, K., Maeda, A., Maeda, J., and Makino, S. (2000) J. Virol. 74,

8127–813414. Kuo, L., and Masters, P. S. (2002) J. Virol. 76, 4987–499915. Corse, E., and Machamer, C. E. (2000) J. Virol. 74, 4319–432616. Baudoux, P., Carrat, C., Besnardeau, L., Charley, B., and Laude, H. (1998)

J. Virol. 72, 8636–864317. Bos, E. C., Luytjes, W., van der Meulen, H. V., Koerten, H. K., and Spaan,

W. J. (1996) Virology 218, 52–6018. Vennema, H., Godeke, G. J., Rossen, J. W., Voorhout, W. F., Horzinek,

M. C., Opstelten, D. J., and Rottier, P. J. (1996) EMBO J. 15, 2020–202819. Laviada, M. D., Videgain, S. P., Moreno, L., Alonso, F., Enjuanes, L., and

Escribano, J. M. (1990) Virus Res. 16, 247–25420. To, L. T., Bernard, S., and Lantier, I. (1991) Vet. Microbiol. 29, 361–36821. Jacobse-Geels, H. E., and Horzinek, M. C. (1983) J. Gen. Virol. 64,

1859–186622. Tooze, J., Tooze, S., and Warren, G. (1984) Eur. J. Cell Biol. 33, 281–29323. Tooze, J., and Tooze, S. A. (1985) Eur. J. Cell Biol. 37, 203–21224. Krijnse-Locker, J., Ericsson,M., Rottier, P. J., andGriffiths, G. (1994) J. Cell

Biol. 124, 55–7025. de Haan, C. A., Kuo, L., Masters, P. S., Vennema, H., and Rottier, P. J.

(1998) J. Virol. 72, 6838–685026. Escors, D., Ortego, J., Laude, H., and Enjuanes, L. (2001) J. Virol. 75,

1312–132427. Ma, H. C., Fang, C. P., Hsieh, Y. C., Chen, S. C., Li, H. C., and Lo, S. Y.

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by guest on August 21, 2015

http://ww

w.jbc.org/

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nloaded from

(2008) J. Biomed. Sci. 15, 301–31028. Huang, Y., Yang, Z. Y., Kong, W. P., and Nabel, G. J. (2004) J. Virol. 78,

12557–1256529. Siu, Y. L., Teoh, K. T., Lo, J., Chan, C.M., Kien, F., Escriou, N., Tsao, S.W.,

Nicholls, J. M., Altmeyer, R., Peiris, J. S., Bruzzone, R., and Nal, B. (2008)J. Virol. 82, 11318–11330

30. Hatakeyama, S.,Matsuoka, Y., Ueshiba, H., Komatsu,N., Itoh, K., Shichijo,S., Kanai, T., Fukushi, M., Ishida, I., Kirikae, T., Sasazuki, T., andMiyoshi-Akiyama, T. (2008) Virology 380, 99–108

31. Bai, B., Hu,Q., Hu,H., Zhou, P., Shi, Z.,Meng, J., Lu, B., Huang, Y.,Mao, P.,and Wang, H. (2008) PLoS ONE 3, e2685

32. Hsieh, P. K., Chang, S. C., Huang, C. C., Lee, T. T., Hsiao, C.W., Kou, Y.H.,Chen, I. Y., Chang, C. K., Huang, T.H., andChang,M. F. (2005) J. Virol. 79,13848–13855

33. Pulford, D. J., and Britton, P. (1991) Virus Res. 18, 203–21734. Nal, B., Chan, C., Kien, F., Siu, L., Tse, J., Chu, K., Kam, J., Staropoli, I.,

Crescenzo-Chaigne, B., Escriou, N., van der Werf, S., Yuen, K. Y., andAltmeyer, R. (2005) J. Gen. Virol. 86, 1423–1434

35. Vo, D., Kern, A., Traggiai, E., Eickmann, M., Stadler, K., Lanzavecchia, A.,and Becker, S. (2006) FEBS Lett. 580, 968–973

36. Sambrook, J., and Russell, D. W. (2001)Molecular Cloning: A LaboratoryManual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Har-bor, NY

37. Wang, C. T., Lai, H. Y., and Yang, C. C. (1999) J. Med. Virol. 59, 180–18838. Wang, S. M., Chang, Y. F., Chen, Y. M., andWang, C. T. (2008) J. Biomed.

Sci. 15, 719–72939. Page, K. A., Landau, N. R., and Littman, D. R. (1990) J. Virol. 64,

5270–527640. Wang, S. M., and Wang, C. T. (2009) Virology 388, 112–12041. Luo, H., Wu, D., Shen, C., Chen, K., Shen, X., and Jiang, H. (2006) Int.

J. Biochem. Cell Biol. 38, 589–59942. Ono, A., and Freed, E. O. (2001) Proc. Natl. Acad. Sci. U.S.A. 98,

13925–13930

43. Sun, X., and Whittaker, G. R. (2003) J. Virol. 77, 12543–1255144. Scheiffele, P., Rietveld, A., Wilk, T., and Simons, K. (1999) J. Biol. Chem.

274, 2038–204445. Narayanan, K., and Makino, S. (2001) J. Virol. 75, 9059–906746. Narayanan, K., Chen, C. J., Maeda, J., and Makino, S. (2003) J. Virol. 77,

2922–292747. Anderson, R. G. (1998) Annu. Rev. Biochem. 67, 199–22548. Choi, K. S., Aizaki, H., and Lai, M. M. (2005) J. Virol. 79, 9862–987149. Li, G. M., Li, Y. G., Yamate, M., Li, S. M., and Ikuta, K. (2007) Microbes

Infect. 9, 96–10250. Baudoux, P., Besnardeau, L., Carrat, C., Rottier, P., Charley, B., and Laude,

H. (1998) Adv. Exp. Med. Biol. 440, 377–38651. Chan, C. M., Ma, C. W., Chan, W. Y., and Chan, H. Y. (2007) Arch. Bio-

chem. Biophys. 459, 197–20752. Lai, C.W., Chan, Z. R., Yang, D. G., Lo,W.H., Lai, Y. K., Chang,M. D., and

Hu, Y. C. (2006) FEBS Lett. 580, 3829–383453. Siu, K. L., Kok, K. H., Ng, M. H., Poon, V. K., Yuen, K. Y., Zheng, B. J., and

Jin, D. Y. (2009) J. Biol. Chem. 284, 16202–1620954. Bishop, G. A. (2004) Nat. Rev. Immunol. 4, 775–78655. Spiegel,M., Pichlmair, A.,Martínez-Sobrido, L., Cros, J., García-Sastre, A.,

Haller, O., and Weber, F. (2005) J. Virol. 79, 2079–208656. Cheung, C. Y., Poon, L. L., Ng, I. H., Luk, W., Sia, S. F., Wu, M. H., Chan,

K. H., Yuen, K. Y., Gordon, S., Guan, Y., and Peiris, J. S. (2005) J. Virol. 79,7819–7826

57. Machamer, C. E., and Rose, J. K. (1987) J. Cell Biol. 105, 1205–121458. Machamer, C. E., Grim, M. G., Esquela, A., Chung, S. W., Rolls, M., Ryan,

K., and Swift, A. M. (1993)Mol. Biol. Cell 4, 695–70459. Swift, A. M., and Machamer, C. E. (1991) J. Cell Biol. 115, 19–3060. Armstrong, J., Patel, S., and Riddle, P. (1990) J. Cell Sci. 95, 191–19761. Locker, J. K., Klumperman, J., Oorschot, V., Horzinek, M. C., Geuze, H. J.,

and Rottier, P. J. (1994) J. Biol. Chem. 269, 28263–2826962. de Haan, C. A., Vennema, H., and Rottier, P. J. (2000) J. Virol. 74,

4967–4978

SARS-CoV M Assembly

12872 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 17 • APRIL 23, 2010

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and Chin-Tien WangChiangHuang, Amber I-Ru Lee, Chien-Cheng

Ying-Tzu Tseng, Shiu-Mei Wang, Kuo-Jung  Syndrome Coronavirus Membrane ProteinSelf-assembly of Severe Acute RespiratoryProtein Synthesis and Degradation:

doi: 10.1074/jbc.M109.030270 originally published online February 12, 20102010, 285:12862-12872.J. Biol. Chem. 

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